In this proposal, we focus on a bacterial transcription regulator GabR that activates the GABA shunt pathway, which has been studied both as an alternative route for nitrogen metabolism and as a pathway related to stress response, including pH homeostasis. Although not essential for survival, the prospect of novel antimicrobial/antivirulence methods via disrupting the stress response invites further investigation on the prevalent GABA shunt pathway in bacteria and its biological functions. In stark contrast to GabT or GabD, GabR is a unique bacterial protein with no functional homologs in eukaryotes, making it a potentially very attractive antimicrobial target. We have recently reported the first crystal structure of full-length GabR as both apo and PLP- bound structures which reveal a head-to-tail GabR dimer prompting our hypothesis that effector-triggered conformational changes enable the recruitment of RNA polymerase complex executing the GabR-dependent transcription activation. We propose to determine how GabR employs both effectors PLP and GABA to trigger transcription activation and to determine how GabR-DNA interactions execute both auto-repression and transcription activation using our experience in protein crystallography and medicinal chemistry as well as established spectroscopic, circular dichroism, florescence anisotropy and biological assays to gain mechanistic and structural insights of GabR-effector interaction, PLP-GABA interaction/reaction and GabR-DNA interaction. Mutagenesis of key residues in combination with medicinal chemistry-guided synthesis of effector mimetics with agonist and antagonist properties will enable the elucidation of molecular mechanisms of GabR, combined with small Angle X-ray Scattering (SAXS). Further, through the design and synthesis of GABA and GABA-PLP analogs for GabR binding and activation, and guided by X-ray crystallography and docking experiments, we aim to disrupt the pH homeostasis of Bacillus subtilis through designing super agonistic ligands of GabR. We will characterize the biological effects of the designed ligands with an eye for super-agonistic compounds for GabR-dependent regulation in our structure- based ligand design, and the potency and biological effects of designed ligands will be characterized with binding assays and assays of transcription activation, and through mechanistic crystallography. Education of both undergraduate and graduate students will be enhanced through the work described in this grant, consistent with the vigorous, demonstrated track record of both PI and co-PI in enhancing and promoting education, particularly involving minorities and underserved populations.